Method and apparatus for particle counting



Aug. 11, 1970 3, KLlNG E'TAL METHOD AND APPARATUS FOR PARTICLECOUNTING Filed Jan. 5, 1966 INVENTORS. NELSON a KLING KENTHNEGERSMITH ATTORNEY United States atent Office U.S. Cl. 356-39 9 Claims ABSTRACT OF THE DISCLOSURE Apparatus and method are described for counting particles suspended in a liquid sample, e.g. red and white cells in a blood sample. The liquid samples are supplied as a flowing stream and divided into two quotient streams which are independently treated for the analysis of a different constituent. The treated increments in each quotient stream are directed successively through a flowcell whereby each successive increment washes the flowcell of contaminants from a previous increment. In the preferred embodiment, treated increments from the quotient streams are directed atlernately and cyclically through the flowcell in opposite directions whereby the flowcell is reversed-flushed by each successive increment.

This invention relates to an improved metod and apparatus for counting the number of particles suspended in a fluid, and more particularly, to the automatic counting of the number of red and white blood cells contained in each of a plurality of sequentially supplied blood samples.

In the U.S. patent application of Jack Isreeli, Ser. No. 427,593, filed Jan. 25, 1965, and assigned to a common assignee, there is disclosed an apparatus which is adapted to the automatic counting of the number of either red or white blood cells contained in each of a plurality of sequentially supplied blood samples. Each of the blood samples is diluted and passed in one direction through a flow cell for counting. Between successive blood samples, a segment of a wash liquid is passed through the flow cell in the opposite direction to wash out the flow cell. During this wash cycle,-no sample counting can proceed. Thus, to provide both red and white cell counts for each of a plurality of blood samples, two procedures are available with the Isreeli system. In one procedure, a portion of each blood sample is off-taken sequentially, and the red (or white) cells are counted; subsequently another portion of each blood sample is off-taken sequentially, and the white (or red) cells are counted. In this procedure, which uses only one flow cell, about 60 counts per hour are made. In the other procedure, two treatment manifolds and flow cells are utilized, one set for independently treating and counting red cells, the other for independently treating and counting white cells. Each blood sample is off-taken sequentially and a portion of each sample is transmitted to the red cell counting set while another portion of each sample is concurrently transmitted to the white cell counting set. In this latter procedure, 120 counts per hour are made, but, as noted, this requires two sets of treatment manifolds and flow cells.

Accordingly, it is an object of this invention to provide a method and an apparatus wherein both red and white cells are automatically counted for each blood sample by a treatment manifold system requiring a single flow cell, in the same time previously utilized by systems requiring two flow cells.

A feature of this invention is a method of red and white cell substantially contemporaneous counting by transmitting blood samples sequentially as an initial flowing stream; continuously dividing the initial stream into two quotient streams, an increment of each quotient stream being a fractional portion of a respective sample from the initial stream; continuously treating one of the quotient streams for red cell counting, and continuously treating the other of the quotient streams for white cell counting; passing sequential increments from one of the quotient streams through a flow cell in one direction, and, in alternation with such increments from the one quotient streams, passing sequential increments from the other of the quotient streams through the flow cell in the other direction.

Another feature of this invention is an appartus comprising a supply means for supplying blood samples sequentially as an initial flowing stream; means for continuously dividing the initial stream into two quotient streams, an increment of each quotient stream being a fractional portion of a respective sample from the initial stream; means for continuously treating one of the quotient streams for red cell counting, and continuously treating the other of the quotient streams for white cell counting; a flow cell; means for detecting and counting the passage of blood cells through the flow cell; and means for passing sequential increments from one of the quotient streams through the flow cell in one direction, and, in alternation with such increments from the one quotient stream, passing sequential increments from the other of the quotient streams through the flow cell in the other direction.

These and other objects, features and advantages of this invention will be apparent from the following disclosure taken in conjunction with the accompanying drawings in which:

FIG. 1 is a pictorial view of an automatic system for counting red and white blood cells in a plurality of blood samples, showing the system at a disposition in which it is counting red cells; and

FIG. 2 is a detail of the system of FIG. 1, showing the system at a disposition in which it is counting white cells.

As shown in the drawing, the blood samples are respectively contained in a plurality of sample containers 10, each of which containers is supported in an indexible turntable 12. The turntable may be part of a sample supply apparatus 13 such as is disclosed in U.S. Pat. No. 3,134,263 issued to Edward B. M. DeJong, on May 26, 1964. A sample take-01f device 14, having a take-01f tube or crook 16, is positioned adjacent the periphery of the turntable, with the crock disposable into and out of each sample container as such container is presented to the take-01f device by the turntable.

The downstream end of the take-off tube 16 is coupled to a first leg of a three-legged junction 18, whose second leg is coupled to a pump tube 20, and whose third leg is coupled to a pump tube 22. These pump tubes are disposed in a peristaltic type pump 24, which may be of the type disclosed in U.S. Pat. No. 2,935,028, issued to Andres Ferrari, Jr. et al., on May 3, 1960. The pump 24 includes a plurality of rollers 26 which are supported by endless chains 28 above a platen, and which successively engage, occlude progressively longitudinally, and release the pump tubes which are disposed between the rollers and the platen. By this arrangement, the blood samples are sequentially aspirated by the take-01f tube 16, and transmitted as an initial flowing stream of samples to the fitting 18. At this fitting the initial stream is continuously divided into a first quotient stream passing through the pump tube 20 and a second quotient stream passing through the pump tube 22.

Advantageously, the sample supply apparatus 13 may have a wash liquid container 30 disposed laterally of the turntable into which the take off tube 16 may be disposed between sequential insertions into the sample containers. As the take oif tube moves between successive sample containers, it will take in air, and if disposed in a wash container, it will also take in wash liquid between successive blood samples. These segments of air and wash liquid white cell counting.

The inlet of a pump tube 32 is coupled to a source 33 of saline diluent, and the inlet of a pump tube 34 is coupled to a source 35 of a relatively inert gas, such as the atmosphere. The outlets of the tubes 32 and 34 are respectively coupled to two legs of a three legged junction 36 whose third leg is coupled by a conduit 38 to the first leg of a three legged junction 40. The outlet of the pump tube 22 is coupled to the second leg of the junction 40 whose third leg is coupled to the inlet of a mixing coil 42. The mixing coil 42 may be of the type shown in the US. Pat. No. 2,933,293 to Andres Ferrari, Jr., issued on April 19, 1960. The air provided by the tube 34 serves to insure the uniform mixing of the sample with the diluent. The outlet of the mixing coil 42 is coupled to a conduit 44 which is coupled to one leg of a three legged junction 46. The second leg of the junction 46 is coupled to the inlet of a pump tube 48, and the third leg is coupled to a conduit 49 which discharges to wash. The inlet of a pump tube 50 is also coupled to the source 33 of saline diluent, and the inlet of a pump tube 52 is coupled to a source 54 of inert gas. The outlets of the tubes 50 and 52 are respectively coupled to two legs of a three legged junction 56 whose third leg is coupled by a conduit 58 to the first leg of a three legged junction 60. The outlet of the pump tube 48 is coupled to the second leg of the junction 60 whose third leg is coupled to the inlet of a mixing coil 62. The outlet of the coil is coupled to a conduit 64. By this arrangement, the quotient stream in the pump tube 22 has been highly diluted in preparation for red cell counting. Since the ratio of red cells to white cells in normal blood is in the order of 20,000 to 1, the white cells in this quotient stream may be disregarded as the counting error produced there-by is negligible.

The inlet of a pump tube is coupled to a source 72 of acetic acid and tergitol, and the inlet of a pump tube 74 is coupled to a source 76 of an inert gas. The outlets of the tubes 70 and 74 are respectively coupled to two legs of a three legged junction 78 whose third leg is coupled by a conduit 80 to the first leg of a three legged junction 82. The outlet of the pump tube 20 is coupled to the second leg of the junction 82, whose third leg is coupled to the inlet of a mixing coil 84. The outlet of the coil is coupled to a conduit 86. By this arrangement, the red cells are hemolyzed while leaving white cells intact and diluted.

The detecting assembly includes a flow cell 92 and a two position, five port valve 94. As shown in U .S. patent application S.N. 427,593, supra, the flow cell has a flat, narrow passageway 96 therethrough, and a photoelectric system, here shown in rudimentary form as a light source 98 and a detector 100, for detecting the passage of a particle, such as a cell, through the passageway. The signals generated by the detector 100 are transmitted to a count/rate circuit 102 which controls the excursion of the stylus of a chart record 104.

The passageway 96 is quite narrow and there is a tendency for debris to be caught therein, which might cause contamination of successive samples. To preclude such contamination, the red cell samples are transmitted through the passageway 96 in one direction, and the white cell samples are transmitted through the passageway in the other direction, whereby the leading portion of each sample reverse flushes the flow cell passageway.

The valve body has five ports 112, 114, 116, 11 8, and 120. The port 114 is coupled by a conduit 122 to the upper end 96U of the passage 96. The port 118 is coupled by a conduit 124 to the lower end 96L of the passageway 96. The port 116 is coupled by a conduit 126 to a pulse damping chamber 128 which is coupled to the inlet of a pump tube 130 whose outlet discharges to waste. The port 112 is coupled by a conduit 132 to the lower leg of an F junction 134, whose middle leg is coupled to the red cell conduit 64, and whose upper, overflow leg is coupled to a conduit 136 which discharges to waste. The port 120 is coupled by a conduit 138 to the lower leg of an F junction 140 whose middle leg is coupled to the white cell conduit 86, and whose upper, overflow leg is coupled to a conduit 142 which discharges to waste.

The valve stem 144 has two conduits 146 and 147. In the first or red cell count position shown in FIG. 1, the conduit 146 interconnectes the ports 112 and 114, while the conduit 147 interconnects the ports 116 and 118. In the second or white cell count position shown in FIG. 2, the conduit 146 interconnects the ports 114 and 116, while the conduit 147 interconnects the ports 118 and 120.

A programmer 148 is electrically coupled to a rotary, two directional solenoid 150, which solenoid is mechani-. cally coupled to the valve stem 144. The count/rate circuit includes two alternative sensitivity controls 152, one for red cell counting one for white cell counting, and the programmer 148 is electrically coupled to switch one or the other into operation. The programmer 148 is also coupled to the drive mechanism of the sample supply apparatus 13.

The programmer 148 cyclically operates the sample supply apparatus to transmit a blood sample to the junction 18 for a given interval, e.g. 60 seconds. The programmer, each half cycle operates the rotary valve 150 to turn the valve stem to its first position for one half of the given interval, e.g. 30 seconds, while the red count sensitivity control is operative; and to its second position for another half of the given interval, e.g. 30 seconds, while the white count sensitivity control is operative.

When the valve stem is in its first position, the red cell sample portion flows through the conduits 64 and 122, through the fiow cell passageway 96, and through the conduits 124 and 126 to the pulse chamber 128 and thence to waste. The white cell sample portion concurrently flows through the conduits 86 and 142 to waste. When the valve stem is in its second position, the white cell sample portion flows through the conduits 86, 138, and 124, through the fiow cell passageway 96, through the conduits 122 and 126 to the pulse chamber 128 and thence to waste. The red cell sample portion concurrently flows through the conduits 64 and 136 to waste. It will be appreciated that the leading part of each sample portion which flows through the flow cell passageway reverse flushes out contaminants left from the immediately preceding sample portion, while the remaining part of such sample portion provides an accurate cell count, which is detected and recorded by the detecting assembly 90 and the recorder 104 as shown in US. Patent Application S.N. 427,593, supra.

If the wash reservoir 30 is utilized, it may be supplied by a source 200 of saline solution coupled to the inlet of a pump tube 202. In such an event, the interval of time that the wash liquid flows through the off-take tube must be accommodated for by the programmer.

While a preferred embodiment of this invention has been shown and described, it will be appreciated that other embodiments will become apparent to those skilled in the art upon reading this disclosure, and, therefore, the invention is not to be limited to the disclosed embodiment, except as required by the hereto appended claims.

What is claimed is:

1. A method of counting red and white cells substantially contemporaneously in each of a plurality of blood samples, said method comprising: transmitting the blood samples sequentially as an initial flowing stream; continuously dividing the initial stream into two quotient streams, an increment of each quotient stream being a fractional portion of a respective sample from the initial stream; continuously treating one of the quotient streams for red cell counting, and continuously treating the other of the quotient streams for white cell counting; pumping sequential increments from one of the quotient streams through a flow cell in a first direction and, in alternation with such increments from the one quotient stream, pumping sequential increments from the other of the quotient streams through the flow cell in the opposite direction from said first direction; and counting the cells in each of the increments as it passes through the flow cell.

2. A method of counting cells according to claim 1 wherein the steps of pumping comprise sequentially and cyclically pumping increments from one of the quotient streams in a first direction through the flow cell and increments from the other of the quotient streams in a second direction through the flow cell which is opposite to the first direction.

3. A method of analysis of a plurality of fluid samples, said method comprising: pumping each of the samples sequentially through a flow cell, each successive sample being pumped through the flow cell in a direction opposite to that of the immediately preceding sample, whereby the flow cell is reverse' flushed by each sample; and analyzing each sample as it passes through the flow cell.

4. A method of analysis to claim 3 which includes: transmitting the samples sequentially as an initial flowing stream; and continuously treating the samples for analysis before passing the samples through the flow cell.

5. A method of analysis according to claim 3 which includes: transmitting the samples sequentially as an initial flowing stream, continuously dividing the initial stream into a plurality of quotient streams, an increment of each quotient stream being a fractional portion of a respective sample from the initial stream; continuously treating each of the quotient streams for analysis for a different characteristic; sequentially and cyclically pumping an increment from each of the quotient streams through the flow cell, each successive increment being passed through the flow cell in a direction opposite to that of the immediately preceding increment, whereby the flow cell is reverse flushed by each increment, and analyzing each increment as it passes through the flow cell.

6. Apparatus for counting red and white cells substantially contemporaneously in each of a plurality of blood samples, said apparatus comprising: supply means for the blood samples; a flow cell; means for forming an initial flowing stream of each sample in succession; means for counting the red and white blood cells as they pass through said flow cell; means for dividing each initial stream of each sample into two quotient streams; means for treating at least a portion of one of said quotient streams for the counting of red cells and for treating at least a portion of the other of said quotient streams for the counting of red cells; and means connected to said treating means for alternately pumping portions of said treated quotient streams through said flow cell in opposite directions for the counting of the red and white cells by said counting means.

7. Apparatus for the analysis of a plurality of samples, said apparatus comprising: means for supplying the samples sequentially; a flow cell; and pumping means coupled to said supply means for pumping a first portion of each sample through the flow cell in a first direction and for pumping a second portion of each sample through the flow cell in a second direction opposite to said first direction, means for treating each of said first and second portions for different constituents to be analyzed, and means for analyzing said first and second portions during passage through said flow cell.

8. Apparatus according to claim 7 wherein said supply means comprises means for supplying said samples sequentially as an initial flowing stream, said pumping means includes means for continuously dividing the initial stream into two quotient streams, said quotient streams comprising said first and second portions, respectively, of each sequential sample in said initial stream, and said pumping means including means for directing said first portions in one of the quotient streams through said flow cell in said first direction and, in alternation therewith, directing said second portions in the other of the quotient streams through said flow cell in said second direction.

9. Apparatus according to claim-7 wherein said pumping means includes means for cyclically and alternately directing first and second portions of each sample through said flow cell.

References Cited UNITED STATES PATENTS 851,388 4/1907 Wallace 2l0411 893,070 7/1908 Gobbi 210411 1,945,839 2/1934 Von Maltitz 210411 3,047,367 7/ 1962 Kessler 23-230 3,165,693 1/ 1965 Isreeli et a1. 3,225,645 12/1965 Baruch et al 250218 3,241,432 3/1966 Skeggs et al. 8814 RONALD L. WIBERT, Primary Examiner W. A. SKLAR, Assistant Examiner US. Cl. X.R. 

